A novel promising strain of Trichoderma evansii (WF-3) for extracellular α-galactosidase production by utilizing different carbon sources under optimized culture conditions.

Chauhan A, Siddiqi NJ, Sharma B - Biomed Res Int (2014)

Bottom Line:
The incubation time, temperature, and pH for the maximum enzyme synthesis were found to be 120 h (5 days), 28°C, and 4.5-5.5, respectively.Among the metal ions tested, Hg was found to be the strongest inhibitor of the enzyme.Among the chelators, EDTA acted as stronger inhibitor than succinic acid.

Affiliation: Department of Biochemistry, Faculty of Science, University of Allahabad, Allahabad 211002, India.

ABSTRACTA potential fungal strain of Trichoderma sp. (WF-3) was isolated and selected for the production of α-galactosidase. Optimum conditions for mycelial growth and enzyme induction were determined. Basal media selected for the growth of fungal isolate containing different carbon sources like guar gum (GG), soya bean meal (SM), and wheat straw (WS) and combinations of these carbon substrates with basic sugars like galactose and sucrose were used to monitor their effects on α-galactosidase production. The results of this study indicated that galactose and sucrose enhanced the enzyme activity in guar gum (GG) and wheat straw (WS). Maximum α-galactosidase production (213.63 U mL(-1)) was obtained when the basic medium containing GG is supplemented with galactose (5 mg/mL). However, the presence of galactose and sucrose alone in the growth media shows no effect. Soya meal alone was able to support T. evansii to produce maximum enzyme activity (170.36 U mL(-1)). The incubation time, temperature, and pH for the maximum enzyme synthesis were found to be 120 h (5 days), 28°C, and 4.5-5.5, respectively. All the carbon sources tested exhibited maximum enzyme production at 10 mg/mL concentration. Among the metal ions tested, Hg was found to be the strongest inhibitor of the enzyme. Among the chelators, EDTA acted as stronger inhibitor than succinic acid.

fig2: Effect of different carbon sources on the level of α-Gal activity (U/mL) secreted by T. evansii (WF-3) up to 11 days. GG = guar gum, SM = soya bean meal, WS = wheat straw, GAL = galactose, and SUC = sucrose. The experiments have been conducted three times and the values are shown as mean ± SD. Blank bar = not detected (for first day enzyme activity).

Mentions:
In order to find out the optimum incubation time for maximum enzyme production, α-Gal activity was determined in the culture filtrate after interval of every 24 h. The media containing different substrates like GG, SM, and WS and also in combination with galactose and sucrose were incubated at 28°C in shaking flask (120 rpm). The effect of different incubation periods on α-Gal production using basal fermentation medium is shown in Figure 2. The optimum production was obtained at the fifth and sixth day of incubation period; maximum activity was shown by WF-3 fungal strain into the culture filtrate (213.63 U/mL) in media containing GG + GAL followed by media containing SM (170.36 U/mL) and others. However, the media containing GG + SUC and WS + SUC registered minimum enzyme activity on days 5 and 6. The longer incubation of up to 11 days showed decreasing trend in the enzyme activity (Figure 2). It has already been established that the microbial production of α-Gal varies with the growth rate [35] as the activity increases with increase in biomass concentration [39]. Similar observations have been reported by Anisha and Prema [40], where they have shown that the biomass increased with the increasing period of incubation with the increase in enzyme production. The highest enzyme production for AGP47 and AGP42 was reported after fourth and sixth day of incubation, respectively, after which cell mass declined and also the enzyme production [40]. Similar to our results, El-Gindy et al. [41] reported that the sixth day of incubation was the best for the experimental fungi where A. awamori produced maximum α-Gal activity (2.172 U/g), while A. carbonarius reached maximum α-Gal production at incubation period of 6 days (2.280 U/g). The activity of α-Gal secreted by A. awamori and A. carbonarius showed reducing trend, the values being 1.6 U/mL and 1.8 U/mL, respectively, upon increasing duration of incubation after 7 days. The decline of total enzyme activity could be considered to be the result of inhibition of cellular functions and due to depletion of nutritional factors from the growth medium or deactivation of enzyme due to pH change or due to inducer exclusion or due to action of secreted fungal proteases.

fig2: Effect of different carbon sources on the level of α-Gal activity (U/mL) secreted by T. evansii (WF-3) up to 11 days. GG = guar gum, SM = soya bean meal, WS = wheat straw, GAL = galactose, and SUC = sucrose. The experiments have been conducted three times and the values are shown as mean ± SD. Blank bar = not detected (for first day enzyme activity).

Mentions:
In order to find out the optimum incubation time for maximum enzyme production, α-Gal activity was determined in the culture filtrate after interval of every 24 h. The media containing different substrates like GG, SM, and WS and also in combination with galactose and sucrose were incubated at 28°C in shaking flask (120 rpm). The effect of different incubation periods on α-Gal production using basal fermentation medium is shown in Figure 2. The optimum production was obtained at the fifth and sixth day of incubation period; maximum activity was shown by WF-3 fungal strain into the culture filtrate (213.63 U/mL) in media containing GG + GAL followed by media containing SM (170.36 U/mL) and others. However, the media containing GG + SUC and WS + SUC registered minimum enzyme activity on days 5 and 6. The longer incubation of up to 11 days showed decreasing trend in the enzyme activity (Figure 2). It has already been established that the microbial production of α-Gal varies with the growth rate [35] as the activity increases with increase in biomass concentration [39]. Similar observations have been reported by Anisha and Prema [40], where they have shown that the biomass increased with the increasing period of incubation with the increase in enzyme production. The highest enzyme production for AGP47 and AGP42 was reported after fourth and sixth day of incubation, respectively, after which cell mass declined and also the enzyme production [40]. Similar to our results, El-Gindy et al. [41] reported that the sixth day of incubation was the best for the experimental fungi where A. awamori produced maximum α-Gal activity (2.172 U/g), while A. carbonarius reached maximum α-Gal production at incubation period of 6 days (2.280 U/g). The activity of α-Gal secreted by A. awamori and A. carbonarius showed reducing trend, the values being 1.6 U/mL and 1.8 U/mL, respectively, upon increasing duration of incubation after 7 days. The decline of total enzyme activity could be considered to be the result of inhibition of cellular functions and due to depletion of nutritional factors from the growth medium or deactivation of enzyme due to pH change or due to inducer exclusion or due to action of secreted fungal proteases.

Bottom Line:
The incubation time, temperature, and pH for the maximum enzyme synthesis were found to be 120 h (5 days), 28°C, and 4.5-5.5, respectively.Among the metal ions tested, Hg was found to be the strongest inhibitor of the enzyme.Among the chelators, EDTA acted as stronger inhibitor than succinic acid.

Affiliation:
Department of Biochemistry, Faculty of Science, University of Allahabad, Allahabad 211002, India.

ABSTRACTA potential fungal strain of Trichoderma sp. (WF-3) was isolated and selected for the production of α-galactosidase. Optimum conditions for mycelial growth and enzyme induction were determined. Basal media selected for the growth of fungal isolate containing different carbon sources like guar gum (GG), soya bean meal (SM), and wheat straw (WS) and combinations of these carbon substrates with basic sugars like galactose and sucrose were used to monitor their effects on α-galactosidase production. The results of this study indicated that galactose and sucrose enhanced the enzyme activity in guar gum (GG) and wheat straw (WS). Maximum α-galactosidase production (213.63 U mL(-1)) was obtained when the basic medium containing GG is supplemented with galactose (5 mg/mL). However, the presence of galactose and sucrose alone in the growth media shows no effect. Soya meal alone was able to support T. evansii to produce maximum enzyme activity (170.36 U mL(-1)). The incubation time, temperature, and pH for the maximum enzyme synthesis were found to be 120 h (5 days), 28°C, and 4.5-5.5, respectively. All the carbon sources tested exhibited maximum enzyme production at 10 mg/mL concentration. Among the metal ions tested, Hg was found to be the strongest inhibitor of the enzyme. Among the chelators, EDTA acted as stronger inhibitor than succinic acid.